16S-Seq of nuclease-free water blank run through Qiagen QIAamp PowerFecal DNA Isolation Kit
The V4 region of the 16S rRNA gene was amplified using the 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT) primers. The forward primers contained the common sequence linker tag 1 (CS1, ACACTGACGACATGGTTCTACA) and the reverse primers contained the common sequence linker tag 2 (CS2, TACGGTAGCAGA-GACTTGGTCT) (Moonsamy et al 2013). Amplicons were generated using a two-stage targeted amplicon sequencing approach (Bybee et al 2011), and the resulting amplicons sequenced on the Illumina MiSeq platform (Caporaso 2012). The first stage was performed in 96-well plates (10 L reactions) using MyTaq HS 2X mas-termix (Bioline, London, UK) and the following thermal cycling conditions: 95C for 5 minutes, 28 cycles of 95C for 30 seconds, 55C for 45 seconds and 72C for 30 seconds. The second stage was performed in 96-well plates (20 L reactions), also utilizing MyTaq HS 2X mas-termix. In each well a unique primer pair with a 10-base barcode was used. These were obtained from Access Array Barcode Library for Illumina (Fluidigm, South San Francisco, CA, USA). The following thermal cycling conditions were used for this second stage: 95C for 5 minutes, 8 cycles of 95C for 30 seconds, 60C for 30 seconds, and 72C for 30 seconds. A final 7-minute elongation step was performed at 72C.